Academic journal article Environmental Health Perspectives

Human Cell Exposure Assays of Bacillus Thuringiensis Commercial Insecticides: Production of Bacillus Cereus-Like Cytolytic Effects from Outgrowth of Spores

Academic journal article Environmental Health Perspectives

Human Cell Exposure Assays of Bacillus Thuringiensis Commercial Insecticides: Production of Bacillus Cereus-Like Cytolytic Effects from Outgrowth of Spores

Article excerpt

Most contemporary bioinsecticides are derived from scaled-up cultures of Bacillus thuringiensis subspecies israelensis (Bti) and kurstaki (Btk), whose particulate fractions contain mostly B. thuringiensis spores ([is greater than] [10.sup.12]/L) and proteinaceous aggregates, including crystal-like parasporal inclusion bodies (PIB). Based on concerns over relatedness to B. cereus-group pathogens, we conducted extensive testing of B. thuringiensis (BT) products and their subfractions using seven human cell types. The Bti/Btk products generated nonspecific cytotoxicities involving loss in bioreduction, cell rounding, blebbing and detachment, degradation of immunodetectable proteins, and cytolysis. Their threshold dose (Dt [approximately equals] 5 x [10.sup.-14]% BT product/target cell) equated to a single spore and a target cell half-life ([tLD.sub.50]) of approximately 16 hr. At Dts [is greater than] [10.sup.4], the [tLD.sub.50] rapidly shifted to [is less than] 4 hr; with antibiotic present, no component, including PIB-related [Delta]-endotoxins, was cytolytic up to an equivalent of approximately [10.sup.9] Dt. The cytolytic agent(s) within the Bti/Btk-vegetative cell exoprotein (VCP) pool is an early spore outgrowth product identical to that of B. cereus and acting possibly by arresting protein synthesis. No cytolytic effects were seen with VCP from B. subtilis and Escherichia coli. These data, including recent epidemiologic work indicate that spore-containing BT products have an inherent capacity to lyse human cells in free and interactive forms and may also act as immune sensitizers. To critically impact at the whole body level, the exposure outcome would have to be an uncontrolled infection arising from intake of Btk/Bti spores. For humans, such a condition would be rare, arising possibly in equally rare exposure scenarios involving large doses of spores and individuals with weak or impaired microbe-clearance capacities and/or immune response systems. Key words: biopesticides, bioreduction, cell death, cytolytic factors, endotoxin, immunodetection, immune sensitization, [sup.35]S-methionine, vegetative cells. Environ Health Perspect 108:919-930 (2000). [Online 18 August 2000] http://ehpnet1.niehs.nih.gov/docs/2000/108p919-930tayabali/abstract.html

The major sources of microbe-based biotechnology products (MBPs) released into the environment today are commercial Bacillus thuringiensis (BT) products that are derived from similarly produced, large-scale, sporulation-phase cultures of B. thuringiensis subspecies, mainly, israelensis (Bti) for targeting the larval stage of blood-sucking flies (Dipterans) and kurstaki (Btk) for targeting foliage-eating larvae of moths (Lepidoptera) (1,2). The combined production of these BT products exceeds 500 metric tons annually in North America (3). From the point of view of homogeneity, these biopesticides (also known as bioinsecticides, biorationale pesticides, or biological control agents) are very complex (2). However, whether in dry (powdered) or liquid states, they are very similar because they are essentially mixtures of culture ingredients that include, in increasing order of their mass, variable amounts of minerals, extracellular nucleic acid, a large spectrum of proteins (mostly sporulation phase-specific), and viable spores, often exceeding [10.sup.12]/L of BT product (2,4,5). The liquid versions of both Bti- and Btk-derived commercial BT products can be easily fractionated by differential centrifugation to yield similar-sized particulate fractions containing [is greater than] 99.9% of the spores and also the proteinaceous component made up of both regular and irregular amorphic structures (2). This proteinaceous material cannot be quantitatively separated from spores and other culture debris. The regular variably sized aggregates are predominantly those that are often referred to as crystal toxins (1,6). These structures make up the bulk of the mature parasporal inclusion body (PIB) matrix and contain most of the [Delta]-endotoxins precursors (also any partially processed or degraded forms) encoded by specific cry genes (1,6). …

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